Within eukaryotic cells, genome duplication initiates at multiple sites on each chromosome. Replication initiation events in diploid mitotic cells proceed in a precise order and are strictly regulated by a series of cell cycle checkpoint signaling pathways. Some of these regulatory constraints, however, are often relaxed in cancer cells. Because the processes that coordinate replication ultimately converge on chromatin, understanding the molecular events that precede DNA replication at the chromatin level is crucial if we are to fully understand cell growth. Critical information about this process is missing because protein complexes that initiate chromosomal replication seem to bind DNA indiscriminately. To gain a complete understanding of the DNA replication process we must resolve how this non-specific DNA binding translates into highly coordinated replication. Our studies are based on the hypothesis that sequence-specific signaling molecules associate with replication initiation sites on chromatin where they modulate the local activity of the ubiquitous replication machinery and dictate both the location and timing of replication initiation events. To test this hypothesis, we characterize protein-DNA interactions at replication initiation sites and identify interactions that play regulatory roles in the DNA replication process. We use two approaches to characterize DNA-protein interactions at replication initiation sites. The first approach utilizes distinct DNA sequences, termed replicators, which facilitate the initiation of DNA replication. We have initially identified these replicator sequences and we now use them as bait to isolate protein complexes that potentially regulate replication. In recent studies we have identified two discrete DNA-protein complexes within one replicator element. One of these complexes includes chromatin remodeling proteins that determine both replication timing and transcriptional activity (Mol Cell Biol. 31:3472-84). Another complex includes RepID, a member of the DDB1-Cul4-associated-factor (DCAF) family, which binds a subset of replication initiation sites and is required for replication at those sites. Our studies have demonstrated that RepID associates with chromatin-loop interactions between a replicator element and a distal regulatory sequence within the human beta globin (HBB) locus. This year, we have developed the methodology to characterize RepID interactions with other proteins, identified RepID protein partners using a non-biased approach and pinpointed protein domains within RepID that facilitate DNA-protein and protein-protein interactions. The second approach involves developing tools to map replication initiation sites throughout the genome, and using these tools to analyze DNA replication in the context of chromatin modifications and transcriptional activity. The developed methods involve massively parallel sequencing and single-fiber imaging of replication fork progression. These procedures allow us to study the dynamics of DNA replication at the whole-genome level. Using this methodology we can test whether groups of replication initiation sites share specific properties - for example, if they associate with a particular chromatin feature. We can also identify groups of initiation sites that respond in a similar fashion to a cellular challenge, and test whether distinct groups of replication initiation sites are regulated through association with particular proteins (such as RepID). Our recent studies generated a comprehensive dataset of replication initiation sites for several human cancer cell lines (Genome Res. 21:1822-32). We also identified a positive correlation between replication initiation and CpG methylation, and a negative correlation between replication and high levels of transcription. We also used genome-wide data to identify DNA and histone modifications that associate with replication initiation events. We also used genome-wide data to identify DNA and histone modifications that associate with replication initiation events. For example, we observed strong associations between replication initiation and both DNAse hypersensitive sites and dimethylated histone H3 lysine 79, which exhibits a dynamic cell cycle distribution (PLoS Genet. 9:e1003542). This year, we have also performed collaborative studies demonstrating that replication timing patterns correlate with global patterns of replication initiation sites (PLoS Genet. 9:e1003542). Concomitantly, we participated in a collaborative theoretical simulation study demonstrating that the locations of replication initiation sites could provide a sufficient framework for determination of replication timing, and no special replication timing program was required Mol Syst Biol. 10:722). We plan to continue our combined studies at the local and whole genome level to identify proteins that modify chromatin or modulate distal interactions to determine replication initiation sites and dictate replication timing. We previously observed that mild exposure to replication inhibitors decelerate replication via mechanisms that involve the cancer-predisposing proteins BLM helicase, Mus81 nuclease, and ATR kinase (J Mol Biol. 375: 1152). We have recently observed that Mus81 endonuclease activity also affects the normal pace of DNA replication and the frequency of replication initiation during unchallenged growth. In contrast, ELG1/ATAD5, which is also involved in cellular responses to perturbed replication, did not affect replication initiation rates (J Cell Biol. 200:31). These results imply that enzymes previously thought to be DNA repair specialists may participate in surveillance mechanisms that regulate DNA replication during unperturbed growth. In other collaborative studies, we have demonstrated that the retinoblastoma/E2F pathway plays a role in the regulation of replication patterns during murine development (Mol Cell Biol. 34:2833). In the future we will investigate how protein-DNA interactions that are required for DNA replication are modulated in response to environmental challenges and anti-cancer drugs.